There is little doubt that we are in the midst of a worldwide epidemic of diabetes. Almost 16 million people in the US are thought to be afflicted, a third of whom are undiagnosed. Insulin resistance is recognized as a characteristic trait of the disease, defined by the inability to respond to normal circulating levels of insulin. The primary lesion in this state involves defects in the uptake and storage of glucose in muscle and fat cells. Targeting these defects holds the key to the development of new therapeutic approaches. However, understanding the specific lesions that cause insulin resistance in patients with type 2 diabetes will first require a better grasp of the cell biology of insulin action. To this end, the molecular events involved in the regulation of glucose uptake by insulin will be investigated, with special attention to the underlying basis for the specificity of actions of the hormone. Recent data has uncovered a new pathway involving the activation of two related G proteins called TC10-alpha and -beta. These proteins are both localized to discrete domains of the plasma membrane called lipid rafts. Aim 1 will evaluate the differences in the activation of these proteins by insulin, focusing on their regulation by guanyl nucleotide exchange factors, inactivation by GAP proteins, their effects on the actin cytoskeleton and the stimulation of Glut4 vesicle trafficking to the plasma membrane. In Aim 2 we will complete the targeted disruption of the TC10-beta gene, and initiate the disruption of the gene encoding TC10-alpha. We will then cross these knockout mice to evaluate the role of this pathway in the regulation of glucose homeostasis. Aim 3 will focus on the effectors downstream of TC100-beta, following on preliminary data implicating three candidate proteins in the regulation of the actin cytoskeleton. Finally, the downstream effectors of TC10-alpha will be studied in Aim 4, with special attention to a newly described signaling pathway involving the activation of the atypical protein kinases C, via the molecular scaffolding protein Par3 and Par6. Together, these approaches will allow for the evaluation of the importance of this novel pathway in insulin action, setting the stage for future investigations into its potential role in the development of diabetes.